Study of Antimicrobial Activity of Black Cumin Seeds (Nigella Sativa L

S al & urg ic ic d a e l

M P f a o Utami, et al., J Med Surg Pathol 2016, 1:3

l h Journal of Medical & Surgical

o DOI: 10.4172/2472-4971.1000127 r

y o

J Pathology ISSN: 2472-4971

Research Article Open Access Study of Antimicrobial Activity of Black Cumin Seeds (Nigella sativa L.) Against Salmonella typhi In Vitro Amalia Tri Utami1*, Bogi Pratomo2 and Noorhamdani3 1Medical Program Study, Brawijaya University, Malang, Indonesia 2Internal Medicine Laboratory, Saiful Anwar General Hospital, Malang, Indonesia 3Microbiology, Brawijaya University, Malang, Indonesia *Corresponding author: Amalia Tri Utami, Medical Program Study, Brawijaya University, Malang, Indonesia, Tel: +628813477409; E-mail: [email protected] Received date: April 05, 2016; Accepted date: May 12, 2016; Published date: May 19, 2016 Copyright: © 2016 Utami AT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Objective: To determine the effectiveness of extracts of black cumin seeds (Nigella sativa L.) as an antimicrobial against Salmonella typhi in vitro. And can determine minimum inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC) from extracts of black cumin seeds (Nigella sativa L.) against Salmonella typhi.

Design: This experimental study used post-test only control group design with four time repetition. Step one was cultivating bacteria in liquid medium with various concentration of extract, that was 40%, 42.5%, 45%, 47.5%, 50% with two control, extract control and bacterial control.

Results: The MIC (Minimal Inhibition Concentration) is 45% concentration of extract. Step two was plating in NAP (Nutrient Agar Plate) medium. The MBC (Minimal Bactericidal Concentration) is 47.5% concentration of extract. The result of experiment was knew there are different average of Salmonella typhi colony from every group. The result experiment was analyzed by One Way Anova Test. The hypothesis test of MBC show significant differentiation, and then was continued with regression test. The conclusion of this study was the addition of extract concentration occurred reduction average of Salmonella typhi colony.

Keywords: Antimicrobial; Extract ethanol; Nigella sativa L.; subsequent development of resistance of Salmonella typhi, several Salmonella typhi; In vitro countries reported a strain of multi-drug resistance (MDR) Salmonella typhi that is resistant to two or more antibiotics commonly used are Introduction ampicillin, chloramphenicol and cotrimoxazole. The development of MDR Salmonella typhi so fast in some countries resulting in mortality Bacterial of the genus Salmonella are capable of causing a large cases of typhoid fever in children increases, the experts seek another number of human infections, including typhoid fever, systemic alternative treatment for typhoid fever that fever down quickly, shorter infections, septicemia, and gastroenteritis varying clinically from treatment times and reduced relapse [4]. watery diarrhoea to dysentery [1]. The continued development of drug resistance of microorganisms to Typhoid fever is caused by microorganisms Salmonella enterica other microorganisms one that makes health organizations in the subspecies enterica serotype typhi (abbreviated as Salmonella typhi), is world were moved. Food Drugs Association (FDA) one of them, a systemic infectious disease, and is still a health problem in Indonesia. implementing an action plan against drug resistance with 4 stages, Endemic infectious diseases are growing in Asia, Africa, Latin namely, surveillance, development of a new drug product effective and America, the Caribbean and Oceania, including Indonesia, where natural, education, and research on resistance. The fourth stage is sanitation is poor. Although WHO declared typhoid fever not already underway FDA since 8 years ago [5]. including deadly diseases such as pneumonia, tuberculosis, malaria, measles and HIV/AIDS, but the epidemic of typhoid fever can be like Currently in Indonesia is developing a new paradigm in the field of in 1989, where 11 countries stricken with typhoid fever. In that year health, namely the use of natural herbs and traditional medicines. 10% of patients who died of typhoid fever. WHO said there were 20 Medicinal plants as natural resources have not been exploited and million deaths each year are caused by typhoid fever. WHO reported developed in depth is still very open to be researched and developed incidence of typhoid fever increased by 15.41% in 2008 [2]. Young under on finding an effective drug as an anti-microbial, especially in adult patients often experience severe complications such as bleeding typhoid fever. The use of herbal medicines is expected to be an and perforation of the bowel which often end in death [3]. Because of alternative for the community to prevent the side effects of antibiotic the high morbidity and mortality rates of typhoid fever, the various use [6]. Therefore, the need for alternatives to overcome these parties seek to resolve this problem. problems, logical, scientifically appropriate methods of "scientific knowledge". One possible approach is to use plants (herbs) that have In the last five years, clinicians in several countries observe any known or are still in the form of alleged anti-microbial effects that can cases of typhoid fever are severe and even fatal child, which was caused be used widely, with a good level of security or no side effects [7]. by Salmonella typhi strains resistant to chloramphenicol. In the

J Med Surg Pathol, an open access journal Volume 1 • Issue 3 • 1000127 ISSN:2472-4971 Citation: Utami AT, Pratomo B, Noorhamdani (2016) Study of Antimicrobial Activity of Black Cumin Seeds (Nigella sativa L.) Against Salmonella typhi In Vitro. J Med Surg Pathol 1: 127. doi:10.4172/2472-4971.1000127

Page 2 of 4 10.4172/jmsp.1000127 Since one century, the Greek physician, Dioscorides, it has been bacterial growth while at a concentration of 47.5% is not contained doi: reported that black cumin or Nigella sativa or Black Seed to treat a bacterial growth. variety of ailments such as headaches, nasal congestion, dental pain, Based on this, researchers took concentration from 40% to 50% to and illness internist. During the golden age of Islam, by Al Biruni, the determine the value of MIC and MBC. The final concentration used in seeds of Nigella sativa used nutritional ingredients. In the treatment in this study is 40%, 42.5%, 45%, 47.5% and 50%. Researchers over the Greco-Arab or Greek-Tibb, from Hippocrates, Galen, and Avicenna, difference in the concentration range close up with the aim of MIC and Nigella sativa is a very valuable remedy in treating gastrointestinal MBC values obtained are more accurate. dysfunction and hepatitis described as a stimulant for the different conditions [8]. Religious factors also increase the confidence of his people to take advantage of Nigella sativa. In the hadith of Prophet The minimum inhibitory concentration (MIC) growth of Muhammad SAW said that black cumin can cure all diseases except Salmonella typhi death. Various studies show that in Nigella sativa contains essential oils Solution between black cumin seed extract and bacterial extracts (volatile oil). Essential oils are generally anti-microbial, anti- were made with a concentration of 40%, 42.5%, 45%, 47.5%, and 50% inflammatory [9]. Antimicrobials of black cumin seed extract in the for the then incubated at 37°C for 18-24 hours, then observed the form of active compounds such as: alkaloids, saponins, tannins, change of turbidity turbidity which is later will be able to know the melantin, derivatives terpenoids, and timokinon [7]. So far, studies level of minimum inhibitory concentration (MIC) black cumin seed have been conducted on the effects of Nigella sativa strains of the extract on the growth of Salmonella typhi. bacterium Vibrio colera and E. coli. The research that published in the journal Pharmaceutical Pakistan showed that more potent volatile oils to kill both bacteria compared antibiotics such as ampicillin and tetracycline [9].

Methodology Research conducted experimental research laboratory is using the tube dilution method to determine the concentration of extract of black cumin seeds (Nigella sativa L.), which can affect the growth of the bacteria Salmonella typhi isolates in vitro. The tube dilution method is to determine the MIC and MBC. This method includes two phases, phase of testing materials/extracts against isolates of Salmonella typhi in the media brain heart infusion broth to determine the MIC and the stage of planting in the medium Nutrient To Plate to determine MBC. There are five groups of treatment, coupled with germ control (0% extract material) and material control extract (100% Figure 1: Dilution results for Test Tube MIC against Salmonella extract material), with four repetitions. typhi.

Result Shown in the Figure 1 that at a dose of 40%, 42.5%, is still turbid media, while at a dose of 45% compared with the media already clear Extract ethanol of Nigella sativa L. KK. KHM then obtained at a dose 45%. The end result of black cumin seed extract liquid is obtained black. From Figure 1 it can be seen that at the extract concentration of 0% Then extract that has obtained a suspension made by bacteria test BC (Bacterial Control) with four repetitions showed turbidity levels are (Salmonella typhi) that are mixed CMC 0.1% (Cetyl Methyl Cellulose) very murky. At a concentration of 40% extract showed turbidity levels as emulsifier between the distilled water to extract oil that there are are very murky. In the extract concentration of 42.5% showed turbidity elements, the extract concentration of 40%, 42.5%, 45 %, 47.5% and levels are murky. At a concentration of extract of 45, 47.5%, 50%, and 50%. As a control material is 100% concentration is the concentration 100% (Material Control) showed clear. Based on observations of in which the untreated bacteria (pure extract), while the concentration turbidity levels in BHI media can be seen MIC value is the of 0% is a bacterium that controls the concentration of the extract is concentration of the extract 45%. The concentration meaning that 45% not given at all. The concentration of each treatment was obtained is the lowest concentration of an antimicrobial that is able to inhibit using the formula dilution with reference based on exploratory study the growth of bacteria Salmonella typhi. (preliminary investigation) has been done before. Exploratory research using a concentration of 2.5%, 5%, 10%, 20%, The minimum bactericidal concentration (MBC) Salmonella 40%, and 50% by the tube dilution method using serial dilutions. After typhi planting the bacteria in the medium NAP, the exploration results that Once known the value of MIC of observations in liquid media, then range from 50% already do not grow bacteria, while at a concentration planting the NAP with streaking method. These observations were of 40% was obtained bacterial colonies. Then the researchers explored made by counting the number of Salmonella typhi colonies that grow back with a concentration from 40% to 50%, using a concentration of on the medium NAP after planting one ose of each concentration to 40%, 42.5%, 45%, 47.5% and 50%. After planting the bacteria in the determine the antimicrobial effect of black cumin seed extract on the medium NAP was found that the concentration of 45% is still a growth of Salmonella typhi. KBM value obtained by counting the number of colonies on the plate using a colony counters "LAB-LINE".

doi: 10.4172/jmsp.1000127 Page 3 of 4

In this study, the dose of the extract obtained KBM 47.5%. At this dose obtained colony growth. At the bacteria control the growth of bacteria plate, there was no growth of colonies on the plate into four. The obtained as much as 8.4 x 107 colonies. following image (Figure 2) on the medium NAP streaking agar plate:

Figure 3: Mean colony of Salmonella typhi each deuteronomy inter Figure 2: Results Salmonella typhi streaking on NA medium for treatment. MBC test Caption a) Control of germs b) Dose extract 40% c) Dose extract 42.5% d) Dose extract 45% e) Dose extract 47.5% f) Dose extract 50%. When the number of colonies of each treatment concentration compared to the number of colonies of bacteria control, seen a significant reduction of the number of bacterial colonies. Minimum It appears that at a concentration of 40%, 42.5% and 45%, there are Bactericidal Concentration (MBC) is the lowest concentration of still colonies of bacteria grow that amount> 0.1% of the original extract that is capable of killing bacteria, so MBC from black cumin inoculum, at a concentration of 47.5% while there was no growth of seed extract in this study is the extract concentration of 47.5% due to colonies (colony growth of germs <0.1% of colonies the original the concentration had not obtained colony growth on medium NAP inoculum). That is 47.5% doses of the MBC black cumin seed extract. (the growth of bacteria colonies <0.1% of the original colony Then done counting the number of colonies of Salmonella typhi at inoculum). each concentration with four repetitions by using Colony Counter. Counting the number of colonies of Salmonella typhi at each NAP. Discussion Results planting NAP media of various concentrations of the extract Essential oil of black cumin seeds contains active compounds, can be seen in Table 1 and Figure 3 below. namely; alkaloids, saponins, tannins, terpenoids and derivatives. Treatment Repetition Mean Alkaloid class of black cumin seeds are alkaloids isochinolin (nigellimin and nigellimin-N-oxide) and pyrazol alkaloids (nigellidin 1 2 3 4 and nigellicin). Derivatives terpenoids (carvacrol, carvone, limonene,

0% 8.5 x 107 8.4 x 107 8.9 x 107 7.8 x 107 8.4 x 107 4-terpineol, citronellol). Active substances such as anti-microbial function [7]. Alkaloids have a mechanism to interact with bacterial 40% 6.2 x 107 6.4 x 107 6.5 x 107 5.8 x 107 6.2 x 107 DNA by putting themselves between strands of DNA, so that it will interfere with DNA work in organizing all activities of bacteria that 7 7 7 7 7 42.5% 5.1 x 10 4.3 x 10 5.0 x 10 4.2 x 10 4.5 x 10 cause bacterial lysis [10]. Saponin works by damaging the cytoplasmic 45% 1.6 x 107 1.1 x 107 2.9 x 107 1.0 x 107 1.25 x 107 membrane and the membrane of bacteria so disturb organelle bacterial cell permeability [11]. Saponins also inhibit the DNA-polymerase so 47.5% 0 0 0 0 0 that it can interfere with bacterial nucleic acid synthesis. Tannins can

50% 0 0 0 0 0 bind a protein which is owned by bacteria, that adhesin and when this happens it can damage the availability of receptors on the cell surface 100% 0 0 0 0 0 of bacteria. Tannin also can form complexes with compounds irreversible proline, a complete protein, which has a bonding effect of Table 1: The Results mean colony of Salmonella typhi to different inhibition of protein synthesis for the formation of cell walls [12]. treatment. Tannins work by pursed wall/membrane of the cell and disrupt the bacterial cell permeability. Through disruption of the permeability, the From Table 1 it can be seen that the decline in the number of cell cannot perform life activities that are stunted or even die. bacterial colonies in line with increasing concentrations of black cumin Terpenoids works by forming a complex with the protein extracellular seed extract. In the extract concentration of 40% obtained average and bacterial cell membrane causing bacterial membrane is damaged. 7 colony growth as much as 6.2 x 10 , the concentration of the extract The mechanism allows one or more ingredients such active 7 contained 42.5% average growth of 4.5 x 10 colony as much as 45% substances in inhibiting or killing the bacteria Salmonella typhi. extract concentrations are the average growth of the colony as much as Inhibition of the growth of Salmonella typhi is observed from the value 7 1:25 x 10 ; and the extract concentration of 47.5% and 50% had not of MIC (minimum inhibitory concentration) and MBC (Minimum bactericidal concentration). In this study found the MIC at 45%, where

10.4172/jmsp.1000127 Page 4 of 4 doi: the results of the first dilution tube showed clarity compared to the Terpenoids are very active in killing the bacteria. Terpenoids can other doses. In the beginning, before incubation, it was found that the destroy bacterial cell membrane through a cluster lipofiliknya. While higher the concentration pattern, the result will be more turbid other active ingredients such as volatile oil and others have also been dilution tube, for mixing extract more. However, after incubation for known to effect in inhibiting the growth of bacteria. 18-24 hours, the state of being turned away, the higher the From other studies that have been conducted, it has been proved concentration, the more lucid colour tube. This is because, during the that Nigella sativa has the effect of anthelmintics against Ascaris suum incubation, the bacteria multiply. At a dose of 40% and 42.5% Goeze at the concentration of 10% [13]. Nigella sativa also has inhibitory effect was not found. Therefore, the result of the dilution antifungal effect on the growth of Aspergillus clavatus Desm at the tube looks cloudy. As with the concentration of 45%, 47.5% and 50%, concentration of 10% [14]. At the end of the writing of this paper the where there is inhibition of growth of bacteria, resulting in a dilution authors hope that this study can provide maximum benefit for all tube visible in the clear. parties who wish to take advantage in it for the good, including for Research on the in vitro test Nigella sativa against Aeromonas further research, and for all parties aware of the essence of herbal hydrophila done by Tumar and Boimin which concluded that the medicine. lowest concentration of Nigella sativa extracted, resulting in inhibition zone against Aeromonas hydrophila in value of 2%. Sedangkan in in Conclussion vitro studies that performed by Salman et al. proved that Nigella sativa extract MIC was 2% against Pseudomonas aeruginosa and 1% against The minimum inhibitory concentration (MIC) extract of black Staphylococcus aureus. This proves that black cumin seed extract is cumin seeds (Nigella sativa L.) of the bacterium Salmonella typhi is effective for gram positive and negative bacteria. obtained at doses 45%. The kill minimal concentration (MBC) extract of black cumin seeds (Nigella sativa L.) of the bacterium Salmonella MBC obtained from the study of 47.5%, which is not found colonies typhi obtained at doses 47.5%. of Salmonella typhi growth at all in order Nutrient Plate or the number of colonies less than 0.1% original inoculum. Means that at a dose of 47.5% is the minimum the concentration of black cumin seed extract References can kill Salmonella typhi. 1. Arsyi IA (2008) Antibacterial activity test Ethyl Acetate Extract Ethanol Fraction Leaves Arbenan (Duchesnea Indica Andr.Focke) against Black cumin seeds has anti-microbial effect is of essential oil content Stapyhlococcus aureus and Pseudomonas aeroginosa multiresistant or volatile oil. The essential oil contains active substances such as from antibiotic along with Thin Layer Chromatography Profile. the class of alkaloids, saponins, tannins, and terpenoids which serves 2. Anggarini P (2009) Antibacterial Effects Skin Extract Pomegranate as an anti-microbial. Alkaloids are alkaloids isochinolin (nigellimin (Punica granatum) against Salmonella typhi In-vitro. and nigellimin-N-oxide) and pyrazol alkaloids (nigellidin and 3. Braundwald E (2005) Harrison’s Principles of Internal Medicine. 16th nigellicin). Derivatives terpenoids (carvacrol, carvone, limonene, 4- (edn.) Philadelphia, McGrawHill. terpineol, citronellol) [7]. 4. Cowan MM (1999) Plant products as antimicrobial agents. Clin Microbiol Rev 12: 564-582. The working mechanism of alkaloids as antimicrobial herein Damayanti SW (2008) Antifungal Study Extract Black Cumin Seed attributed to its ability to berinterkalasi with bacterial DNA by putting 5. (Nigella sativa L) to Aspergillus clavatus Desm Growth. Essay. Biology themselves between strands of DNA [10]. This causes the genetic Major FMIPA. encoding process through DNA transcription and protein translation 6. Ferdous (2006) In vitro antibacterial activity of the volatile oil of Nigella bacteria become impaired and can result in damage to DNA that sativa seeds against multiple drug-resistant isolates of Shigella spp. and bacterial cell lysis. isolates of Vibrio cholerae and Escherichia coli. Phytotherapy Research 6: 137-140. Saponins inhibit DNA polymerase that can interfere with nucleic Finegold SM, Dan Barron EJ (1994) Bailey and Scott’s Diagnostic acid synthesis. Saponins are secondary metabolites that are found in 7. Microbiology. (9th edn.) USA: CV Mosby Company. nature, consisting of sugar groups that bind to the aglycone or 8. Hilman I (2005) Taking Lessons from Habbatus Sauda. sapogenin. 9. Kasno BI (2000) Typhoid fever, Learning Starting From Problems. Tannin as antimicrobial working on complex formation with 10. Katzer G (2006) Nigella sativa. proteins, namely through hydrogen bonding and covalent. Works 11. Tollefson L (2008) FDA's Role in Antimicrobial Resistance. which include the inactivation of microbial adhesion molecules, 12. Lili M, Ahmad FA, Ascobat G, Pratiwi A (2004) Cost Effectiveness enzymes, protein transport through the cell membrane, destruction of Analysis of Typhoid Fever Treatment Children Using chloramphenicol the substrate and bind to the bacteria cell wall polysaccharides [10]. and Ceftriaxone in Fatmawati Hospital Jakarta in 2001-2002 8: 59-64. These compounds form complexes with polysaccharides. Inactivation 13. Mahmudah (2010) Antihelmintic effect of Nigella sativa l. seed to Ascaris adhesin by tannin causing bacteria can not stick to the intestinal suum Goeze in vitro. epithelial cells of the host (human). Terpenoids can destroy bacterial 14. Ronny SV (2008) Typhoid fever, Decides Chain Transmission. cell membrane through a cluster lypofilic.

J Med Surg Pathol, an open access journal Volume 1 • Issue 3 • 1000127 ISSN:2472-4971